Among the plasmids found in P. aeruginosa that contribute to its antibiotic resistance and consequent success as a nosocomial pathogen, those belonging to the P-2 incompatibility group are of particular interest because of their prevalence, ability to mediate resistance to the otherwise effective anti-Pseudomonas drugs amikacin, carbenicillin, gentamicin, and tobramycin, and their recently discovered huge size of 280-312 megadaltons. This proposal is for studies on the physical and genetic organization of P-2 plasmids, which also include the metabolic plasmids CAM and OCT found in soil pseudomonads. A representative P-2 plasmid, pMG5, determining resistance to tobramycin, sulfonamide, mercuric chloride, phenylmercuric acetate, borate, and tellurite, among other markers, has been chosen for detailed study. After endonuclease treatment, restriction fragments will be separated by agarose gel electrophoresis and RPC-5 column chromatography. The order of fragments and sites for additional endonucleases will be established by DNA hybridization using a modification of the Southern technique. Since the number of endonuclease fragments may be too great for resolution directly, attempts will be made to obtain shortened derivatives of pMG5 for analysis. Hybrids between Pseudomonas plasmid RP4 and pMG5 will be selected by an in vivo technique or between Pseudomonas plasmid R1162 and pMG5 by in vitro ligation to provide smaller targets for restriction analysis and information about gene localization. Insertional mutation and deletion formation by transposon Tn7 will be used ot localize pMG5 genes further. Finally, the digestion patterns of other IncP-2 plasmids including CAM and OCT will be compared to determine the relationships within this interesting plasmid group.